Human ITPase (encoded by the ITPA gene) is thought to exclude noncanonical (deoxy)nucleoside triphosphates ((d)NTPs) from (d)NTP pools. Noncanonical purine NTPs arise spontaneously in cells from the purine salvage pathway and oxidative damage of canonical (d)NTPs. The critical role for ITPase orthologs in the sanitization of dNTP pools has been demonstrated in bacteria, yeast and murine systems where ITPase deficiencies result in sensitivity to noncanonical purines, a delay in cell cycle progression and chromosomal abnormalities including double-strand breaks. ITPase deficiency in mice is lethal due to cardiomyopathy which is thought to result from disorganization of sarcomere structure. This indicates that a defect in the maintenance of quality ATP pools leads to a phenotype that is consistent with mouse models of human cardiomyopathy. About 5% of the human population has been identified as having decreased ITPase activity, and ITPA status has been linked to altered outcomes for patients treated with either thiopurines or ribavirin. For patients undergoing thiopurine therapy, toxicity is associated with ITPA polymorphism, but for patients undergoing ribavirin treatment, ITPA polymorphism is associated with improved outcomes. Both of these drugs are prodrugs and are bioactivated to form NTPs. Thiopurine nucleoside triphosphates are substrates for ITPase, and it has been hypothesized that the nucleoside triphosphate form of ribavirin would be a substrate for ITPase. Despite the fact that ITPase has been studied for over 40 years, little is known about how ITPase discriminates between canonical and noncanonical (d)NTPs. This proposed research program aims to investigate the amino acid residues of human ITPase that are important for substrate specificity. We hypothesize that alteration of the amino acids implicated in substrate discrimination will result in an enzyme with altered specificity for noncanonical (d)NTPs. We have previously constructed 10 novel substrate specificity mutants. In this study we identified three mutants with enhanced selectivity for noncanonical (d)NTPs. We rationalize that our specific aims will provide a basis for developing small molecules and gene therapy treatments to modulate ITPase activity in an effort to reverse drug toxicity and provide tools to better understand cardiac function regarding noncanonical purines. The specific aims for this proposal include: (1) directly determine kinetic constants for canonical and noncanonical NTPs with select existing substrate specificity ITPase mutants, (2) perform molecular modeling of substrate specificity for ITPase mutants, and (3) engineer and test additional substrate specificity ITPase mutants based on existing and future data. Realization of our specific aims will provide a basis for developing small molecules and gene therapy treatments to modulate ITPase activity in an effort to reverse drug toxicity, provide tools to better understand cardiac function regarding noncanonical purines, and enhance our understanding of ITPase biochemistry.